The non-segmented negative-strand RNA viruses comprise some of the most serious and uncontrolled human viral pathogens. Against such a group of pathogens there are two effective research strategies: to address each virus individually and develop virus-specific therapeutics or prophylactics, or to select a prototype virus and examine every aspect of its replication cycle in depth, with the goal of developing general concepts for intervention. Both approaches are valid, but the latter generally has more far-reaching long- term consequences. Among the non-segmented negative-strand RNA viruses, the rhabdovirus vesicular stomatitis virus (VSV) has been studied as the prototype for several decades, with the result that it is one of the best-understood animal viruses. The development of methods to engineer changes into the genome of negative-strand RNA viruses at the cDNA level and recover infectious virus revolutionized research on these viruses. It is now possible to investigate the effect of specific mutations on every aspect of the biology of negative-strand RNA viruses. In the past grant period major advances were made in defining the cis-acting signals involved in the processes of RNA transcription and replication. Our goal now is to determine the mechanisms by which these cis-acting elements control transcription and replication, to determine how they interact with trans-acting factors, and to analyze their importance in the viral life cycle. We propose to do this by addressing the following specific aims: 1. To identify residues involved in nucleocapsid protein (N):RNA encapsidation by structure-guided mutagenesis using semi-randomized oligonucleotides introduced into an infectious VSV cDNA and allowing natural selection to determine the range of possible alternative genotypes and phenotypes. 2. To identify residues critical for N protein flexibility and N:N interactions using structure-guided mutations. 3. To investigate the RNA sequence requirements for the encapsidation of specific RNAs. 4. To use the power of natural selection to identify N protein:RNA interacting residues. 5. To analyze the mechanism of polymerase attenuation at gene junctions. 6. To test a model for the mechanism of transcriptional attenuation.